FIG. 4.

Z does not inhibit transcription. (A) Z overexpression does not reduce levels of cyclin D1 transcripts. Total cyclin D1 mRNA levels were determined for cells transfected with the indicated constructs by using Northern analysis. Cyclin D1 mRNA levels were normalized to 28S and 18S rRNA band intensities. Experiments were carried out in duplicate and varied as shown. (B) Z does not alter transcription. CMV immediate-early promoter fragment (Promega) was transcribed in the presence of 30 μM (each) Z, GST, bovine serum albumin, or H₂O. RNA production was monitored by incorporation of [³⁵S]rUTP and autoradiography. The second panel shows the effect of heating extract prior to the assay (H.E.). “Cold” refers to the use of unlabeled rUTP, and “No ex.” means no extract was used. (C) Z does not alter the distribution of cyclin D1 mRNA. RNA distribution in cellular fractions overexpressing the listed constructs was monitored. The percentage of nuclear cyclin D1 (solid bars) or GAPDH (open bars) RNA is given. Values of band intensities in each lane were normalized to band intensities of 28S and 18S rRNA to correct for differences in loading. RNA levels were normalized to the distribution of total RNA in the cell. Experiments were carried in duplicate and varied by the percentage given.